胃蛋白酶和木瓜蛋白酶水解核桃蛋白工艺研究

以核桃粕为原料,以水解度为考察指标,研究胃蛋白酶和木瓜蛋白酶对核桃蛋白的水解作用.结果表明：木瓜蛋白酶为酶解核桃蛋白最佳酶,其最佳酶解条件为底物浓度4％、酶质量分数5％、酶解 pH 值为7．0、酶解温度50℃、酶解时间4 h;在该条件下,木瓜蛋白酶酶解核桃蛋白水解度可高于25．76％.     

甲醇溶液对木瓜蛋白酶催化活性的影响

试验结果表明,木瓜蛋白酶在一定浓度甲醇溶液中水解酪蛋白的活性显著上升;动力学测定表明,该酶在甲醇溶液中Km下降;在甲醇溶液中木瓜蛋白酶催化酪蛋白水解反应的最适pH为8.5,最适温度为70℃;紫外差示光谱研究表明,在甲醇溶液作用下木瓜蛋白酶的二级结构发生了变化;荧光发射光谱研究表明,木瓜蛋白酶在甲醇溶液中发射峰位向低波长移动,荧光峰值明显增高。     

酶水解制备花生粕多肽工艺的优化

目的：优化研究花生粕制备多肽的工艺。方法：采用酶法水解提取多肽、用总蛋白试剂盒（TP）双缩脲比色法在540nm处测定其含量。结果：用木瓜蛋白酶水解花生粕蛋白得到多肽的最佳工艺条件为：加酶量6 300u/g原料、温度45℃、底物浓度10%、酶水解时间5h。     

双酶法制备螺旋藻多肽的工艺研究

选用碱性蛋白酶和木瓜蛋白酶结合的双酶法对螺旋藻蛋白进行水解。其中,对木瓜蛋白酶水解螺旋藻蛋白的工艺进行优化。以水解度为指标,研究了酶解时间、酶与底物比、pH和酶解温度4种因素对酶解反应的影响。在此基础上设计了3因素(加酶量、酶解温度和pH)3水平的响应面试验。结果表明碱性蛋白酶水解螺旋藻蛋白的最佳酶解条件为:加酶量4300 U/g,pH 7.0,酶解温度55℃,酶解时间160 min;木瓜蛋白酶的最佳酶解条件为:酶底比为4.5%,酶解温度60℃,pH 6.5,酶解时间210 min。利用碱性蛋白酶和木瓜蛋白酶结合的双酶法制得的多肽水解度可达32.90%,与单酶法相比,水解度明显提高。     

降胆固醇活性花生多肽制备工艺的研究

以冷榨花生粕为原料,酶解制备具有降胆固醇活性的花生多肽。以体外结合胆酸盐的能力为指标,考察了酶的种类、温度、pH值、加酶量、酶解时间等因素对花生多肽降胆固醇活性的影响,确定最优蛋白酶为木瓜蛋白酶,通过正交试验的方法,确定了酶解花生粕制备降胆固醇活性花生多肽的最佳工艺条件为：温度50℃、pH 7.0、加酶量4 000 U/g底物、酶解时间4 h。     

蛋白质的酶水解过程研究

进行了蛋白质酶水解过程的研究。结果表明木瓜蛋白酶对混合蛋白质的亲和力最强 ,而 1398蛋白酶的亲和力最弱。也表明作用位点和亲和力之间有一定的对应关系 ,Km值和作用位点氨基酸含量比例的相关系数为 0 .90 9。温度影响结果表明温度较低时温度升高加速水解反应过程处主要地位 ;当温度较高时 ,酶失活过程处主导地位。在一定水解时间内的讨论最适温度条件具有更明确的针对性 ,从本研究的采用胰酶 (胰蛋白酶和胰凝乳蛋白酶 )水解 4h的条件下 ,反应温度控制在45～ 5 0℃之间最适     

牦牛骨蛋白的酶解条件研究

以蛋白质水解度为评价指标,辅以固形物溶出率,比较了中性蛋白酶、菠萝蛋白酶和木瓜蛋白酶对牦牛骨蛋白的水解效果,研究了酶用量、料液比(底物浓度)、酶解时间对水解度的影响,采用正交试验对酶解条件进行了优化。结果显示,木瓜蛋白酶是牦牛骨蛋白水解的适宜催化剂。在一定条件下,样品水解度随酶用量和酶解时间的增加而增大,底物浓度过低或过高均不利于原料中蛋白质的酶解。木瓜蛋白酶水解牦牛骨蛋白最佳条件为:酶解温度60℃,酶解时间8 h,酶用量3500 U/g蛋白质,料液比1:25(g:m l)。     

接枝淀粉载体固定化糖化酶的研究

合成了淀粉接枝丙烯腈及丙烯酰胺的两亲性高分子化合物,并以此为载体,用物理吸附方法固定化了糖化酶。最适偶联条件研究表明：缓冲液的浓度,ｐＨ值及吸附时间和加酶量都对固定化酶活力,比活有一定的影响。在最适固定化条件下,固定化酶的活力为１５００Ｕ／ｇ干胶,蛋白载量为２５ｍｇ／ｇ干胶,比活为６０Ｕ／ｍｇ蛋白,比天然酶的比活（８．０Ｕ／ｍｇ蛋白）提高６倍。最适反应温度比天然酶提高１０℃（天然酶最适反应温度为５０℃）．无底物存在下,固定化酶在５５℃的半衰期为２４ｈ,而天然酶只有１ｈ;有底物存在下,固定化酶在５５℃的半衰期为２２０ｈ,４５℃的操作半衰期由外推法算得为６９天,而且该载体对糖化酶有一定的保护作用,当固定化酶在低于５５℃热处理一段时间后,对酶活力有激活作用,酶活力最大可提高４０％。该载体合成简单,固定化方法简单,步骤少,因而为工业上应用提供了一种新的可能性。     

家蚕丙氨酸转氨酶理化性质的研究

丙氨酸转氨酶(ALT)是家蚕丝心蛋白合成中的关键酶,为了阐明丝心蛋白的合成机理及利用ALT活力调控丝物质的形成,有必要深入了解ALT的分子性质。作者在分离纯化家蚕后部丝腺ALT的基础上,应用聚丙烯酰胺凝胶电泳等生化方法进一步研究了该酶的若干理化性质。结果表明,家蚕ALT由两种不同亚基组成,分子量分别是54kd和21kd;最适底物是丙酮酸;最适温度为50℃;最适DH为8．5;钾、钠、钙、镁等离子对酶有激活作用,而锰、铜、锌等离子对酶有抑制作用。研究还表明,该酶是一种依赖于金属离子的酶类,活性中心含有巯基或咪唑基。     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

High-performance affinity chromatography for characterization of human immunoglobulin G digestion with papain

Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) were prepared within the confines of a stainless steel column. Then papain was immobilized on these monoliths either directly or linked by a spacer arm. In a further step, a protein A affinity column was used for the characterization of the digestion products of human immunoglobulin G (IgG) by papain. The results showed that papain immobilized on the monolithic rod through a spacer arm exhibits higher activity for the digestion of human IgG than that without a spacer arm. The apparent Michaelis-Menten kinetic constants of free and immobilized papain, K(m) and V(max), were determined. The digestion conditions of human IgG with free and immobilized papain were optimized. Comparison of the thermal stability of free and immobilized papain showed that the immobilized papain exhibited higher thermal stability than the free enzyme. The half-time of immobilized papain reaches about a week under optimum pH and temperature conditions.     

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED,ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES : I. CHEMICAL PROPERTIES,ACTIVATION-INHIBITION,pH-ACTIVITY,AND TEMPERATURE-ACTIVITY CURVES

1. A study has been made of the properties of a hitherto unreported proteolytic enzyme from the latex of the milkweed, Asclepias speciosa. The new protease has been named asclepain by the authors. 2. The results of chemical, diffusion, and denaturation tests indicate that asclepain is a protein. 3. Like papain, asclepain dots milk and digests most proteins, particularly if they are dissolved in concentrated urea solution. Unlike papain, asclepain did not clot blood. 4. The activation and inhibition phenomena of asclepain resemble those of papain, and seem best explained on the assumption that free sulfhydryl in the enzyme is necessary for proteolytic activity. The sulfhydryl of asclepain appears more labile than that of papain. 5. The measurement of pH-activity curves of asclepain on casein, ovalbumin, hemoglobin, edestin, and ovovitellin showed no definite digestion maxima for most of the undenatured proteins, while in urea solution there were well defined maxima near pH 7.0. Native hemoglobin and ovovitellin were especially undigestible, while native casein was rapidly attacked. 6. Temperature-activity curves were determined for asclepain on hemoglobin, casein, and milk solutions. The optimum temperature was shown to increase with decreasing time of digestion.     

胃蛋白酶水解绿豆分离蛋白的工艺

选用胃蛋白酶对绿豆分离蛋白进行酶法水解,考察了原料预处理条件、pH、温度、底物浓度等对酶解的影响,结果表明：原料预处理最适条件为沸水浴中90℃处理20min,在37℃、pH1．8、底物质量分数7％、酶量6000U／g条件下酶解180min,水解度（DH％）为19．86％,达到了制备小肽的水解度要求。实验证明,经过水解,绿豆分离蛋白各功能特性得到很好的改善。     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

高分子膜载体固定化木瓜蛋白酶

在浸润条件下,以0.5%(v/v)戊二醛交联的高分子膜尼龙载体固定化木瓜蛋白酶。对固定化条件进行了优化,比较了固定化酶与游离酶的酶学参数。结果表明,4℃、pH6.0条件下,将膜载体浸润于2mg/mL酶液中5h,固定化酶活为303.4U/g。固定化酶最适反应pH为6.0～7.0,最适反应温度为65℃。其pH稳定性、热稳定性均比游离酶高。     

Purification and characterization of an aminopeptidase from Mycoplasma salivarium.

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.     

Degradation of the Major Storage Protein of Phaseolus vulgaris during Germination : Role of Endogenous Proteases and Protease Inhibitors

Cotyledons from Phaseolus vulgaris L. (var. Improved Tendergreen) were tested for their activity on α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) and azocasein during a germination periood of 10 days. Both activities increased throughout germination when activity was expressed on the basis of dry weight or protein. That these two activities were most likely due to the action of different enzymes was indicated by the fact that (a) optimal pH for the hydrolysis of BAPNA and azocasein was 8.2 and 5.5, respectively, and (b) the digestion of azocasein was considerably enhanced by mercaptoethanol and partially inhibited by thiol protease inhibitors, N-ethylmaleimide, and E-64, whereas these same regents caused little change in activity toward BAPNA. The three subunits of the major storage protein, G1, disappeared during germination and were accompanied by the accumulation of lower molecular weight products. The breakdown of G1 by extracts of the germinated beans could be demonstrated in vitro at pH 5 to 6. This activity was enhanced by mercaptoethanol and completely abolished by N-ethylmalemide, leupeptin, and E-64. It is concluded that a thiol protease with an acid pH optimum is primarily responsible for the disappearance of the major storage protein during germination. Although an inhibitor of the plant thiol protease, papain, is present in the mature bean and decreases during germination, its role in the control of the breakdown of the storage protein remains to be elucidated.     

Glycosidases of Ehrlich ascites tumor cells and ascitic fluid--purification and substrate specificity of alpha-N-acetylgalactosaminidase and alpha-galactosidase: comparison with coffee bean alpha-galactosidase

Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.     

壳聚糖固定化木瓜蛋白酶的研究

以自制的壳聚糖作为载体,用戊二醛作交联剂,优化了固定化条件,研制成壳聚糖固定化木瓜蛋白酶。其活性回收率达到42—53％,操作半衰期达到一个月以上,对热、乙醇以及尿素的稳定性有很大的提高,Km值为0.67×10~2mg/mL,最适温度65—70℃,最适pH8.0,能使啤酒中的蛋白质浓度从56.5mg/L减少到2.7mg/L,可以消除啤酒的低温混浊现象。